evolution of primary hemostasis in early vertebrates

Evolution of Primary Hemostasis in Early VertebratesSeongcheol Kim, Maira Carrillo, Vrinda Kulkarni, Pudur Jagadeeswaran*

Department of Biological Sciences, University of North Texas, Denton, Texas, United States of America

Abstract

Hemostasis is a defense mechanism which protects the organism in the event of injury to stop bleeding. Recently, weestablished that all the known major mammalian hemostatic factors are conserved in early vertebrates. However, since theirhighly vascularized gills experience high blood pressure and are exposed to the environment, even very small injuries couldbe fatal to fish. Since trypsins are forerunners for coagulation proteases and are expressed by many extrapancreatic cellssuch as endothelial cells and epithelial cells, we hypothesized that trypsin or trypsin-like proteases from gill epithelial cellsmay protect these animals from gill bleeding following injuries. In this paper we identified the release of three differenttrypsins from fish gills into water under stress or injury, which have tenfold greater serine protease activity compared tobovine trypsin. We found that these trypsins activate the thrombocytes and protect the fish from gill bleeding. We found 27protease-activated receptors (PARs) by analyzing zebrafish genome and classified them into five groups, based on tetheringpeptides, and two families, PAR1 and PAR2, based on homologies. We also found a canonical member of PAR2 family, PAR2-21A which is activated more readily by trypsin, and PAR2-21A tethering peptide stops gill bleeding just as trypsin. Thisfinding provides evidence that trypsin cleaves a PAR2 member on thrombocyte surface. In conclusion, we believe that thegills are evolutionarily selected to produce trypsin to activate PAR2 on thrombocyte surface and protect the gills frombleeding. We also speculate that trypsin may also protect the fish from bleeding from other body injuries due to quickcontact with the thrombocytes. Thus, this finding provides evidence for the role of trypsins in primary hemostasis in earlyvertebrates.

Citation: Kim S, Carrillo M, Kulkarni V, Jagadeeswaran P (2009) Evolution of Primary Hemostasis in Early Vertebrates. PLoS ONE 4(12): e8403. doi:10.1371/journal.pone.0008403

Editor: Bruce Riley, Texas A&M University, United States of America

Received September 24, 2009; Accepted November 18, 2009; Published December 23, 2009

Copyright: � 2009 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by a grant from the National Institutes of Health, HL077910 (to PJ). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Hemostasis is a defense mechanism that evolved to protect the

organism in the event of injury to stop bleeding [1]. In mammals,

when a vessel wall is ruptured, the tissue factor on the surface of

the cells in the subendothelial matrix binds to the preexisting

factor VIIa and initiates coagulation by cleaving factor X to Xa,

which then cleaves prothrombin to generate minuscule amounts

of thrombin. This thrombin cleaves factor XI to XIa. XIa then

cleaves factor IX to IXa, which along with cofactor VIIIa, cleaves

larger amounts of factor X to Xa, which then generates explosive

amounts of thrombin from prothrombin. Interestingly, these

reactions occur on the surface of platelets that are anchored to the

subendothelial matrix via collagen and vWF, which themselves

activate the platelets along with the newly generated thrombin.

These events initiate the secretion of ADP, thromboxane and

serotonin, which activate and amplify the aggregation of

platelets.

Recently, we established that almost all the known major

mammalian hemostatic factors are conserved in early vertebrates

[2]. However, since the highly vascularized gills which experience

high blood pressure are exposed to the environment, even very

small injuries could be fatal to fish. Therefore, these vertebrates

must have evolved mechanisms by which they can respond to an

injury or even stress that could cause bleeding in gills. We do not

know whether there are novel mechanisms for activation of

thrombocytes to enhance hemostasis in fish gills.

Trypsins which are mainly produced in pancreas and are

involved in digestion are the evolutionary forerunners for

coagulation proteases [3,4]. These are also expressed by many

extrapancreatic cells such as endothelial cells, epithelial cells,

nervous system and tumor cells [5]. Trypsin, while used mainly for

cleaving extracellular proteins in digestion, also cleaves the

membrane proteins called protease-activated receptors (PARs)

similar to the way thrombin digests PARs on platelet surface and

initiates signaling cascade in enterocytes that are in contact with

trypsin [6,7]. However, nothing is known about the function of

extrapancreatic trypsins and the possible cells that they will be

activating although several studies are suggestive of their role. We

hypothesized that trypsin or trypsin-like proteases from epithelial

cells from organs such as gills may protect these animals from gill

bleeding following injuries. In this paper we identified trypsin in

fish gills and its release under stress or injury into water. We also

found that the trypsin released around the gills activates the

thrombocytes and plays a role in primary hemostasis.

Materials/Methods

All experiments described below were approved by the

Institutional Animal Care and Use Committee.

Chromogenic Assay60 zebrafish (Ekkwell, Gibsonton, FL) maintained under

standard conditions were kept in 150 ml distilled water for 20

PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8403

min. The fish were then removed and water was centrifuged at

7,000 g for 10 min in a Sorvall centrifuge to remove the feces and

other particulates. This particulate free water was then lyophilized

and the dry pellet was dissolved in 200 mL water. This lyophilized

zebrafish water contains proteins and this concentrated protein

solution is called ZW. The protein concentration was estimated by

BioRad Bradford assay kit (Bio-Rad Laboratories Inc., Hercules,

CA) using 10 mL of this ZW. S-2238 activity assay was performed

using 5 mL of ZW in a final assay volume of 200 mL containing 500

mM S-2238 in 50 mM Tris-HCl pH 7.2 ( S-2238 is a thrombin

substrate but trypsin and other serine proteases also cleave this

chromogenic substrate and since it was readily available in our

laboratory we used this substrate). The reaction was incubated for

15 min and yellow color was measured at 405 nm in a 96-well

kinetic microplate reader (Molecular Device Company, Sunny-

vale, CA). For in-gel chromogenic assay, the lane containing the

fish protein was sliced and kept in a small tray and the S-2238

substrate was layered on the gel and incubated for 15 min. The gel

was photographed using a Nikon E995 CoolPix digital camera.

Gill, mouth and nasal washes each was carried out on 200 fishes

by using 10 mL of water and pipetting up and down three times in

these cavities and then samples were pooled, lyophilized and used

in the above chromogenic assays. Animal care was in accordance

with institutional guidelines. Kinetic analysis was performed by

standard methods.

Analysis of ProteinsThe water proteins were resolved on 5–20% premade

polyacrylamide denaturing gel gradient containing 1% SDS

(Bio-Rad Laboratories Inc., Hercules, CA). The samples were

boiled just before loading. The gel was washed to remove SDS and

renatured for 1 hr with renaturation buffer (Bio-Rad Laboratories

Inc., Hercules, CA) before performing the in-gel chromogenic

assay as described above. The gel band that showed positive

activity was sent to the core facility at University of Texas-

Southwestern Medical Center at Dallas for MALDI-TOF

sequencing. For Western blot, ZW was resolved on a 5–20%

SDS-poly acrylamide gradient gel and then transferred to a

nitrocellulose membrane, which was probed with polyclonal rabbit

antisera against human trypsin (PRAHT) and followed by probing

with alkaline phosphatase conjugated (AP) goat anti-rabbit

IgG(Fc). The probed bands were visualized using the AP substrate

BCIP/NBT.

Functional Evaluation AssaysThrombocyte functions were assayed by whole blood aggrega-

tion using a plate tilt assay [8]. Annexin V binding assays were

performed as described earlier [9]. For gill bleeding assay, fish

were placed in 15 ml of 10 mM NaOH solution to induce

bleeding, for 1.5 min with distilled water, 70 ng of ZW, 70 ng of

ZW with 140 ng of PRAHT and 70 ng of ZW with 140 ng of

control IgG. The images taken of the zebrafish were photographed

using a Nikon E995 CoolPix digital camera. The red color pixels

were counted as an index of the area indicating the extent of

bleeding using Adobe photoshop software version 7.0. For calcium

release assay, Xenopus oocytes were microinjected with 50 nl of

PARs mRNA cloned in pSP64T in 10 mM HEPES (pH 7.0) [10].

The oocytes were retained in 16uC for 48 hrs, and then incubated

with 45Ca2+. The oocytes were then washed and a scintillation

count was taken using LC-6000 liquid scintillation counter

(Beckman Coulter, Inc.) at 10 min intervals to determine amount

of calcium released. After 20 min, calcium release was induced

using 5 ng ZW as the agonist. The released 45Ca2+ from oocytes

was measured again at 10 min intervals.

Expression and Activity Analysis of Fish TrypsinsZebrafish trypsin full length cDNAs were generated by RT-

PCR and these cDNAs were cloned into the E.coli expression

vector pMALc2e and after affinity purification and enterokinase

cleavage, trypsin activity was estimated.

ImmunohistochemistryAdult zebrafish was fixed in a fixative, sectioned into 5 micron

thick slices and probed with the anti rabbit antibodies raised

against human trypsin. Secondary antibodies raised against rabbit

with conjugated FITC were used. The images were taken by using

Nikon Eclipse 80 microscope with constant exposure times and

also by using CSU-10 YoKogawa confocal scanner (McBain

Instruments, Simi Valley, CA) coupled to Zeiss 200 M microscope

(Carl Zeiss, Inc. Thornwood, NY).

Results/Discussion

Identification Serine Protease in Zebrafish WaterWe hypothesized that trypsin or trypsin-like proteases from

epithelial cells of organs such as gills may protect these animals

from gill bleeding following injuries. We further hypothesized

that if this activity is found in gills it should be secreted from gills

and should be found in water. To determine the presence of

proteases in fish water we used the serine protease substrate S-

2238 [11]. Zebrafish were housed in fresh water for 20 min, and

then the water was collected, centrifuged to remove debris,

lyophilized, and dissolved in a minimal volume. This zebrafish

water sample contained sufficient enzyme to convert 0.5 ng S-

2238/min/gram of fish using cleavage by human thrombin as the

positive control. This assay yielded reproducible Michaelis-

Menten kinetics with a Km of 0.83 mM and a Vmax of 13 mmol/

mM/mg of ZW (Figure 1A). The sample exhibited a broad range

of pH and temperature dependence. Interestingly, after boiling

and slowly cooling the ZW, the enzymatic activity was almost

completely recovered. In addition, the sample was stable in 2 M

urea; was not altered in the presence of CaCl2; retained partial

activity in 1% SDS; was not inhibited by EDTA (Figure S1),

benzamidine, or PMSF; and was inhibited by leupeptin and

antipain.

Characterization of Protease in ZWTo identify the protease that yielded SCA, the sample was run

on an SDS denaturing gel, and then the gel was layered with S-

2238 following renaturation in order to detect bands containing

substrate cleavage activity. Two bands were identified: one at 55–

75 kDa appearing as a smear and one at 25 kDa (Figure 1B).

These bands were cut out, proteins eluted and analyzed by

MALDI-TOF mass spectrometric sequencing. The 25 kDa band

revealed trypsin peptides corresponding to three different trypsins

(Table S1). In addition, superoxide dismutase and zinc metallo-

protease peptides were detected in this band. Western blotting

with polyclonal rabbit antisera against human trypsin (PRAHT)

revealed three bands corresponding to three trypsins in this band

supporting the data obtained by MALDI-TOF analysis

(Figure 1C). Thus, the SCA resulting from three trypsins is

consistent with the observed multiple peaks for the pH optima

(Figure S1). Also, these results demonstrated that PRAHT is

specific and does not cross react with any other proteases. The 55–

75 kDa band contained the serpins antitrypsin and serpin a1,

proteases such as trypsins and legumain-like protease, and other

proteins unrelated to SCA (Table S2).

Role of Trypsins in Hemostasis


Expression of Protease in TissuesTo test from where the trypsin activity is derived we collected

water after immersing the fish head alone and also immersing the

rest of the body. The water from immersing the head generated

activity whereas the water collected from rest of the body did not

produce trypsins ruling out the skin as a source for trypsin.

Subsequently the gill wash, nasal wash, and mouth wash samples

were analyzed which yielded trypsin activity corresponding to the

25 kDa band, whereas only the samples from the gill wash

corresponded to the larger band. Since water collected after

immersing the fish head alone and then immersing the rest of the

body did not produce trypsins, the skin was not implicated as a

source of these enzymes. In addition, RT-PCR demonstrated that

trypsin mRNA is synthesized in gills, nose, and mouth tissues (Figure

S2). Immunoreactive cells were also detected in the gills and nasal

epithelium (Figure 2). Counterstaining with histamine antibodies

revealed that the trypsin-producing cells are not mast cells [12] (data

not shown). Skin did not show any immunoreactive cells (data not

shown). Confocal microscopy also revealed positive staining in all

the tissues as was observed by the standard microsopy (Figure 3).

Stress Induced Synthesis of Protease ActivityTo investigate whether the secretion of trypsins into water was

enhanced in response to injury and stress, we employed a variety

of stress conditions including crowding, hypoxia, alkaline pH, and

injury by needle prick (Figure 4). Under all of these conditions,

trypsin release into water was significantly higher than from

control fish, suggesting a considerable response to injury or stress.

To determine whether the trypsin is released rapidly, ZW was

examined ten seconds after the needle prick injury. We found

trypsin activity in water in ten seconds. An individual fish on

average produced 0.13 ng of trypsin based on a standard curve

generated from purified bovine trypsin. No aldolase activity was

detected in ZW under these conditions, suggesting that the

increase in trypsin activity is not due to cellular damage or death.

Role of Trypsin in HemostasisEnhancement of trypsin production during stress or injury may

ultimately be beneficial in protecting the organism. In mammals,

stress-induced bleeding occurs in lungs. For example, thorough-

bred horses bleed in their lungs during races, and humans have

bleeding in their lungs under high altitude hypoxic conditions

[13,14]. The non-coagulation proteases may participate in the

breakdown of clotting factors and thus, may regulate extravascular

coagulation [15] and participate in fibrinolysis [16]. This

proteolytic degradation presents a conundrum because if the fish

responds to a bleeding injury by producing more trypsins,

hemostatic defense will be compromised and continued bleeding

will be favored. To decipher the role of trypsins in hemostasis, we

first tested whether the trypsin activity in ZW was inhibited by

PRAHT. Indeed, PRAHT inhibited trypsin activity in ZW

compared to control IgG (Figure S3). Next, we examined the

degree of bleeding following addition of ZW to a zebrafish in

water. Addition of ZW to zebrafish significantly reduced levels of

bleeding upon alkali-induced injury to gills compared to controls

(Figure 5A). Bovine trypsin also reduced bleeding but required

higher concentrations of trypsin compared to ZW (Figure S4 and

Figure 5A). As a corollary experiment, fish in ZW containing

PRAHT exhibited greater bleeding than fish in ZW alone or fish

treated with control IgG. This result indicated that trypsin actually

controls bleeding. Since higher concentration of bovine trypsin

was required to reduce bleeding we suspected that zebrafish

trypsins may be more active than bovine trypsin. Therefore, we

cloned three zebrafish trypsin full length cDNAs as well as the

bovine cDNA into the E.coli expression vector pMALc2e and after

affinity purification and enterokinase cleavage, trypsin activity was

estimated. We found that all three zebrafish trypsins had similar

levels of activities and were tenfold more active than bovine trypsin

per ng of protein (Figure S5). Next, we determined whether

trypsins that are known to activate protease-activated receptors

(PARs) on cells could activate blood cells using the thrombocyte

aggregation assay. In fact, ZW enhanced cellular aggregation in

this assay (Figure 5B), and this activity was inhibited by PRAHT.

Annexin V binding assays, which measure thrombocyte activation,

revealed that the thrombocytes were indeed activated by ZW,

confirming that the observed cellular aggregation was due to

activation of thrombocytes (Figure 5C). Furthermore, this

activation was inhibited by PRAHT (Figure 5C). In both

Figure 1. Protease activity in zebrafish water samples. (A) Michaelis-Menten plot with velocity as a function of [S-2238]. Each sample wasmaintained for 30 min prior to mixing with substrate under the specified conditions. Standard error bars (60.07) were removed to improve clarity ofeach graph. (B) The proteins secreted by zebrafish were separated on a 5–20% Tris-glycine SDS-polyacrylamide gradient gel. The samples loaded inthe lanes are as follows: 1, molecular weight marker (numbers on the left side indicate the size of the band in kDa); 2, 3, ZW; 4, zebrafish mouth wash;5, zebrafish nose wash; and 6, zebrafish gill wash. Lanes 1 and 2 were stained with Coomassie blue dye. Other lanes were stained using 10 mM S-2238following renaturation. Arrows indicate protease activity. (C) Western blot analysis of ZW. Arrows indicate trypsins. The numbers on the left sideindicate the size of the molecular weight markers in kDa.doi:10.1371/journal.pone.0008403.g001



thrombocyte aggregation assay and annexin V binding assay

bovine trypsin activated thrombocytes but not as efficiently as ZW

and required higher concentrations than ZW (Figure 5B, 5C and

Figure S6, S7). Together, these results established that trypsin

present in ZW activates thrombocytes. The level of trypsin

produced by the fish is typically sufficient to initiate thrombocyte

activation.

Hemostatic Defense by Trypsin Involves PAR2To examine the involvement of PARs in this pathway, we tested

whether the Gq inhibitor that blocks the PAR activity would

inhibit the action of trypsin [17]. The Gq inhibitor indeed blocked

the activity of trypsin in ZW (data not shown). Since four

mammalian PARs have been described and PAR2 has been

shown to be preferentially activated by trypsin, we investigated

whether thrombocyte activation by ZW involves PAR2. Since no

information was available for zebrafish PARs, we analyzed the

zebrafish genome and identified 27 PAR-like genes. By sequence

homology, we classified these PARs into two families: PAR1 and

PAR2. These receptors clustered into five groups (three PAR1

groups and two PAR2 groups) based on the tethering peptide

sequences. These signature sequences act as PAR agonists and

suggest extensive gene duplication (Figure 6). Interestingly, four

members (PAR1-21D, PAR1-7C, PAR1-21E, and PAR1-21G)

contain more than two exons while most of the other zebrafish

PARs contain only two exons similar to those found in humans.

Thirteen of these sequences did not contain complete NH2

termini (shown by dotted lines in Figure 6), and therefore, the

tethering peptides were not determined for these members.

According to RT-PCR analyses, PAR1-5A, PAR1-21A, PAR2-

21A, and PAR2-21D were expressed in a pure population of

thrombocytes; however, PAR1-21D expression was not detected

(Figure S8). The full length cDNAs for PAR1-5A, PAR1-21A,

PAR2-21A, and PAR2-21D were cloned into the expression

vector pSP64T to generate translatable RNAs, which were then

injected individually into Xenopus oocytes [10]. After a few hours,

the oocytes were activated by ZW, and the radioactive calcium

release of the oocytes was measured as an index of trypsin

activation of the expressed PARs. PAR2-21A was activated more

than the other PAR members (Figure 5D). In control experiments,

Figure 2. Immunohistochemistry of trypsin in zebrafish. Zebrafish were dissected and sections of mouth, nose, gill, and pancreas were stainedwith H&E (1) and subjected to immunohistochemistry analysis using polyclonal rabbit antisera against human trypsin (PRAHT) (2). Alexa goat anti-rabbit IgG 488 antisera were used as the secondary antibody. Non-immunized rabbit IgG was used in control experiments (images not shown). 20Xlens was used.doi:10.1371/journal.pone.0008403.g002



human thrombin activated PAR1-5A and PAR1-21A but failed to

activate PAR2-21A (Figure S9). These experiments provided

evidence that ZW trypsin proteases activate PAR-2 in thrombo-

cytes and play a significant role in primary hemostasis.

We next synthesized PAR tethering peptides, which mimic

protease cleavage and signaling, in order to determine whether the

peptides induced signaling. In plate-tilt and annexin V binding

assays, the PAR1-21A (PTQQTL) and PAR1-5A (SFSGFF)

peptides yielded moderate thrombocyte activation while the

PAR2-21A (SLVVIQ) peptides resulted in significant thrombocyte

activation (Figure 7). The GFTGEE and TDEQTE peptides failed

to induce activity. In addition, SLVVIQ stopped alkali-induced

gill bleeding and mimicked trypsin activation (Figure 7). Thus,

under stress conditions, fish respond by producing extravascular

trypsin and prevent gill bleeding by activating thrombocytes via

PAR2 signaling.

ConclusionsThis investigation demonstrates the secretion of trypsins mainly

from gills by fishes into the surrounding water in response to injury

and activating thrombocytes, providing evidence for their role in

hemostasis. Since gills are exposed to the outside environment due

to the constant flow of water, and because they are extremely

important in vital respiratory function, gills should be protected

against injury more than any other organ in the body. In fact, the

gill bleeding is a common problem due to toxic ammonia (http://

www.algone.com/fish_poisoning.php). In their natural habitat fish

with wounded gills are commonly noticed. Since fish gills are

highly vascularized and are a point of high blood pressure it is

possible that they are evolutionarily selected to produce trypsin to

protect them from bleeding before even coagulation cascade is

activated. This paper also demonstrates that three trypsins are

secreted and they are more active than bovine tryspin. This

finding raises important questions such as what is the mechanism

of trypsin secretion. Is trypsin stored as trypsinogen and then

cleaved by another enzyme when it is secreted or is trypsin stored

to similar to mast cell tryptases and then released when under

injury [18]. Thus, we predict that this work opens new doors for

future investigations of these studies.

Also, we speculate the trypsin may not be exclusively protecting

the gills from bleeding but may also protect the fish from bleeding

from other body injuries. This protection theoretically stems from

the fact that fish in locomotion produce von Karman Vortex

Street similar to what is produced by torpedoes when they move

[19]. Earlier workers using zebrafish adults and larvae and 20–40

mm polystyrene spheres in particle image velocimetry, established

the flow fields and vortices and their diameters [20]. Based on this

study, any molecule should follow the physical principles of

hydrodynamic motion. As a result of the von Karman Vortices,

even a few molecules of trypsin produced by the gills will quickly

be in close contact with the body. We estimated about 0.13 ng

trypsin is produced during a ten second period from an injured

fish. Taking the vortex core radius as 0.5 cm and the maximum

height of the vortex cylinder (body width of zebrafish) as 0.5 cm,

the core cylinder volume will be about 0.4 ml volume in a given

vortex. Therefore, the concentration of trypsin in the core cylinder

would be 0.325 ng/ml, which is approximately 1/10th of the

circulating VIIa concentration in mammals that is sufficient to

initiate blood clotting when the stream of blood is leaking from the

vessel during injury. In reality, these vortex cylinder heights are

not as big as the body width and are usually small. Similarly, the

amounts of trypsin released may vary depending upon the number

Figure 3. Detection of trypsin by confocal microscopy. Confocal DIC images (a, 60X and b, 100X) of zebrafish sections of mouth, nose, gill andpancreas stained by immunohistochemistry using primary antibody (a) PRAHT, (b) Non-immunized rabbit IgG. Alexa Fluor 488 goat anti-rabbit IgGwas used as secondary antibody. We used two channel image acquisition to examine the expression of trypsins that were probed with Alexa Fluor. A488 nm and a 405 nm laser were used to excite the Alexa Fluor (Green) and autofluorescence (Blue), respectively. Columns 1 and 2 show brightfieldand fluorescence images respectively.doi:10.1371/journal.pone.0008403.g003



of vortices generated per second. Despite these variations, since

fish trypsins are far more active than mammalian trypsins, the

trypsin following the von Karman Streets must be effective in

activating thrombocytes. During thrombosis, thrombus forms even

though the blood is flowing and this blood flow does not wash

away the thrombus (unless the clot is weak). Similarly in gills where

there is laminar flow of water, trypsin is very effective and

subsequently when it exits the gills, it assumes the complex

hydrodynamic flow where the moving vortex of trypsin water will

activate the thrombocytes. Thus, binding of trypsin to the PAR-2,

in order to enhance thrombocyte signaling is not difficult to

conceive in moving vortex and particularly since initial thrombo-

cytes are anchored to the subendothelial surface (via collagen and

von Willebrand factor). Along the body musculature, obviously,

the vortex helps prevent trypsin to be diluted away by the water,

particularly since fish generates suction pressures due to the

vorteces along the fish body. In both gills and on body

musculature, in addition to thrombocyte activation by trypsin

that initiates primary hemostasis, clotting cascade triggered by

VIIa-tissue factor interactions will generate thrombin subsequently

and take over the primary hemostasis to complete the plugging of

the injured area by generation of fibrin (secondary hemostasis). It

should be noted that the body and gills are protected by mucus

and, thus, they are protected from trypsin action similar to the

protection of digestive tract by mucus cells.

In addition to trypsin we also found antitrypsin in our MALDI-

TOF analysis. This finding would make sense because if excess of

trypsin is produced it may be damaging the exposed vessels during

injury. Therefore, production of antitrypsin seems to control the

trypsin activity. However, observed trypsin activity in the complexes

is puzzling. One explanation is that during incubation in gel, trypsin

could dissociate from the complex could give activity. Such release

of complexes has been noted earlier. Interestingly, we noted that the

55–75 kDa band likely appears as a smear. This is because there are

multiple trypsins and serpins along with their possible degradation

products and thus different size complexes are possible. Also, the

complexes seem to be covalently linked because these proteins have

been run on denaturing polyacrylamide gels. This is consistent with

the finding that covalent complexes do form between serpins and

serine proteases [21].

Figure 4. Response of trypsin activity to stresses. (A) To assess the effects of crowding, 10 and 100 zebrafish were kept separately in 400 ml ofwater for 30 min. The water samples (40 ml) were lyophilized and then resuspended in 1 ml distilled water. Resuspended ZW from these two groups(60 ml and 6 ml respectively) were used in S-2238 cleavage assay. (B) The effects of hypoxia were examined in two fish that were kept in 200 ml water(normoxia) and 200 ml 10% oxygen water (hypoxia) for 30 min. The water samples (40 ml) from each condition was lyophilized and thenresuspended in 1 ml distilled water. Resuspended ZW (90 ml) was used in S-2238 cleavage assay. (C) To assess the effects of pH, 50 ml water wasadjusted with NH4OH to either pH 7 or pH 10 and 5 fish were kept in this water for 30 minutes. The water was immediately neutralized with HCl.From these two samples, 90 ml of water were directly used (without lyophilization) in the S-2238 cleavage assay. (D) To examine the effects of injuryon trypsin activity, the head of zebrafish was immersed in 1 ml distilled water in a 1.5 ml microcentrifuge tube. The water samples collected after10 sec from uninjured and tail-injured fish were lyophilized and resuspended in 200 ml distilled water. Resuspended ZW (90 ml) was used in the S-2238cleavage assay.doi:10.1371/journal.pone.0008403.g004



The PARs are susceptible for proteolytic cleavage which sets

the signal transduction in various types of cells including

platelets. In humans there are four PARs and two of them are

expressed in human platelets, PAR1 and PAR4. In mouse

platelets PAR3 and PAR4 seem to play a role in thrombin

signaling. This paper documents that there are 27 PARs which

appear to be products derived from tandemly duplicated genes.

We could only classify them based on the tethering peptides into

two families, PAR1 and PAR2. Thus, PAR3 and PAR4 seem to

have evolved later in evolution. Interestingly PAR1 in fish are

activated by thrombin while PAR2 is activated by trypsin. Thus,

this investigation clearly documents distinct thrombin and

trypsin receptors.

In summary, we report here trypsins are released by fish into

water in response to injury and stress conditions in order to protect

the fish from bleeding. This finding provides evidence for the role

of trypsins in primary hemostasis of early vertebrates.

Supporting Information

Figure S1 Kinetic analysis of the S-2238 cleaving activity in

zebrafish water. (A) Lineweaver Burk plot with V against l/[S]. (B)

Effect of pH on enzymatic activity. MES (50 mM 2-morpholinoetha-

nesulphonic acid, pH 5–7) and BTP (50 mM bis-Tris-propane, pH 7–

9.5) were used to replace Tris-HCl buffer to study the effect of pH. (C–

G) Effect of temperature, SDS, urea, CaCl2, and EDTA on enzymatic

activity, respectively. (B–G) Each sample was kept for 30 min prior to

mixing with substrate under the above specified conditions. Standard

error bars (60.07) were removed to improve clarity of each graph.

Found at: doi:10.1371/journal.pone.0008403.s001 (0.33 MB TIF)

Figure 5. Effects of ZW on gill bleeding, thrombocyte activation, and PAR-induced signaling. (A) The zebrafish were subjected to the gill bleedingassay as described in Materials and Methods with the following conditions: 1, distilled water (DW); 2, 70 ng ZW; 3, 70 ng of ZW with 140 ng of PRAHT; and 4,70 ng of ZW with 140 ng of control IgG. (B) Plate tilt assay measuring thrombocyte function: 1, PBS; 2, 7 ng ZW; 3, 7 ng ZW with 14 ng control IgG;and 4, 7 ngZW with 14 ng PRAHT. Note the aggregated blood in 2 and 3 is more firm than the control blood 1 and blood 4. (C) Measurement of the functional activity ofZW on thrombocytes by annexin V binding. 7 ng of ZW, 14 ng of PRAHT and 14 ng of control IgG were used. The intensity of images from FITC-annexinfluorescence (green) was measured as mean pixels using Adobe Photoshop software version 7.0. (T test, n = 12). (D) ZW-induced 45Ca2+ release from Xenopusoocytes microinjected with PARs mRNA. The data is presented as the mean value and standard error for 12 oocytes for each PAR mRNA.doi:10.1371/journal.pone.0008403.g005



Figure S2 RT-PCR of trypsins in gill, mouth, and nose from

zebrafish. Tissues from each organ were obtained using microscis-

sors. RNA from tissues was isolated using Absolutely RNA prep kit

from Stratagene, Inc. Trypsin primers were designed using MALDI-

TOF data and NCBI database (Forward 59-TCATGCTGAT-

CAAGCTGA-39 Reverse 59-ATCCAGCGCAGAACATG-39).


Figure S3 Effect of PRAHT on S-2238 cleavage activity of ZW.

S-2238 cleavage activity assay was performed using 70 ng of ZW

in a final assay volume of 200 mL containing 500 mM S-2238 in 50

mM Tris-HCl pH 7.2. Control IgG (140 ng) and polyclonal rabbit

anti-human trypsin (PRAHT) antibody (140 ng) were used in this

assay. Inclusion of fourfold excess of PRAHT completely abolished

trypsin activity (data not shown).


Figure S4 Effect of trypsin on gill bleeding. The zebrafish were

subjected to the gill bleeding assay as described in the Methods

with the following conditions: (A) Distilled water (DW), (B) 200 ng

Figure 6. Alignment of amino acids in the PARs. (A and B) The alignment of the amino acids among different groups of PAR1 and PAR2,respectively. The boxed peptides represent the amino acids involved in tethering to activate thrombocytes. The genomic locations are given inparenthesis (Ensembl Danio rerio versions 44.6e, Zv6 and 49.7c, Zv7).doi:10.1371/journal.pone.0008403.g006



Figure 7. Effect of PAR tethering peptides on thrombocyte function. Top panel: Effect of PAR tethering peptides on thrombocyteaggregation. Plate tilt assay measuring thrombocyte function. (A) DAEFRH (control peptide). (B) TDEQTE (PAR1-21D). (C) SFSGFF (PAR1-5A). (D)PTQQTL (PAR1-21A). (E) SLVVIQ (PAR2-21A). (F) GFTGEE (PAR2-21D). Note the aggregated blood in (C), (D) and (E) is firmer than the control blood (A)and blood (B) and (F). 5 ng of each peptide was used. Middle panel: Measurement of functional activity of PAR tethering peptides on thrombocytesby annexin V binding. 5 ng of each tethering peptide was used. The intensity of images from FITC-annexin fluorescence (green) was measured asmean pixels using Adobe Photoshop software version 7.0 (T test, n = 12). Bottom panel: Effect of SLVVIQ tethering peptides on gill bleeding. Thezebrafish were subjected to the gill bleeding assay as described in Materials and Methods with the following conditions: (A) 50 ng of DAEFRH (controlpeptide) and (B) 50 ng of SLVVIQ (PAR2-21A).doi:10.1371/journal.pone.0008403.g007



bovine trypsin, (C) 100 ng of bovine trypsin with 200 ng of

PRAHT, and (D) 200 ng of bovine trypsin with 400 ng of control

IgG.


Figure S5 The purified trypsins were separated on a 5–20%

Tris-glycine SDS-polyacrylamide gradient gel. The values in the

bottom panel represent the S-2238 cleaving activity of 1 ng of each

sample at A405nm and the mean of three independent determina-

tions 6 the standard error.


Figure S6 Effect of bovine trypsin on thrombocyte aggregation.

Plate tilt assay measuring thrombocyte function. (A) PBS. (B) 20 ng

of bovine trypsin. (C) 20 ng of bovine trypsin with 40 ng PRAHT.

(D) 20 ng of bovine trypsin with 40 ng control IgG. Note the

aggregated blood in (B) and (D) is more firm than the control

blood (A) and blood (C).


Figure S7 Measurement of functional activity of bovine trypsin

on thrombocytes by annexin V binding. 20 ng of bovine trypsin,

40 ng of PRAHT and 40 ng of control IgG were used. The

intensity of images from FITC-annexin fluorescence (green) was

measured as mean pixels using Adobe Photoshop software version

7.0 (T test, n = 12).


Figure S8 RT-PCR for detection of the PAR receptors on

thrombocytes. Thrombocytes were collected using nanoject II.

RNA was isolated from the thrombocytes (n = 455) using

Absolutely RNA prep kit from stratagene, Inc. Primers: GpIIb

(Forward 59- CAGCTGGACAGAATGAAGCA-39 Reverse 59-

GGGAGTCAGCCAAGCTGTAG-39), PAR1-21D (Forward 59-

ACCTTGTTGTATCACTGTGT-39 Reverse 59-TTTCTGT-

AATGAGATGAACC-39), PAR1-5A (Forward 59-TTACCTG-

TACTTCTTTCCAA-39 Reverse 59-AAAACTGCAAAAAC-

TGTTAC-39), PAR1-21A (Forward 59-AACAATCTTGTTTT-

TAGTGC-39 Reverse 59-ATGATGATGATGTAGAAGGA-39),

PAR2-21A (Forward 59-GAGATGTGCAAAGTATCAGT-39

Reverse 59-ACGGTTTGATTGTAGAGATA-39), PAR2-21D

(Forward 59-CTCTGTATCTTTACGACCAG-39 Reverse 59-

CACGTTTGACACTATACACA-39).


Figure S9 Effect of thrombin on PAR-induced signaling.

Thrombin-induced 45Ca2+ release from Xenopus oocytes micro-

injected with PARs mRNAs. The data is presented as the mean

value and standard error for 12 oocytes for each PAR mRNA.


Table S1 Peptide sequences identified from MALDI-TOF

analysis of the 25 kDa band as described in Materials and

Methods.

Found at: doi:10.1371/journal.pone.0008403.s010 (0.04 MB

DOC)

Table S2 Peptide sequences identified from MALDI-TOF

analysis of the 55–75 kDa band as described in Materials and

Methods.

Found at: doi:10.1371/journal.pone.0008403.s011 (0.15 MB

DOC)

Acknowledgments

We thank Drs. Art Goven and Gerard O’Donovan for critical reading of

the manuscript, Lala Zafreen and Ryan Carlson for technical help, Satya

Kunapuli for providing Gq inhibitor, and Dr. Douglas Melton for the

pSP64T plasmid.

Author Contributions

Conceived and designed the experiments: PJ. Performed the experiments:

SK MC VK. Analyzed the data: PJ. Contributed reagents/materials/

analysis tools: PJ. Wrote the paper: PJ.

References

1. Jagadeeswaran P, Gregory M, Day K, Cykowski M, Thattaliyath B (2005)

Zebrafish: a genetic model for hemostasis and thrombosis. J Thromb Haemost 3:

46–53.2. Jagadeeswaran P, Kulkarni V, Carrillo M, Kim S (2007) Zebrafish: from

hematology to hydrology. J Thromb Haemost 5 Suppl 1: 300–304.3. Neurath H, Walsh KA, Winter WP (1967) Evolution of structure and function of

proteases. Science 158: 1638–1644.

4. Hanumanthaiah R, Day K, Jagadeeswaran P (2002) Comprehensive analysis ofblood coagulation pathways in teleostei: evolution of coagulation factor genes

and identification of zebrafish factor VIIi. Blood Cells Mol Dis 29: 57–68.5. Koshikawa N, Hasegawa S, Nagashima Y, Mitsuhashi K, Tsubota Y, et al.

(1998) Expression of trypsin by epithelial cells of various tissues, leukocytes, andneurons in human and mouse. Am J Pathol 153: 937–944.

6. Knecht W, Cottrell GS, Amadesi S, Mohlin J, Skaregarde A, et al. (2007)

Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2to induce inflammation and hyperalgesia. J Biol Chem 282: 26089–26100.

7. Coughlin SR (2005) Protease-activated receptors in hemostasis, thrombosis andvascular biology. J Thromb Haemost 3: 1800–1814.

8. Jagadeeswaran P, Sheehan JP, Craig FE, Troyer D (1999) Identification and

characterization of zebrafish thrombocytes. Br J Haematol 107: 731–738.9. Thattaliyath B, Cykowski M, Jagadeeswaran P (2005) Young thrombocytes

initiate the formation of arterial thrombi in zebrafish. Blood 106: 118–124.10. Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991) Molecular cloning of a

functional thrombin receptor reveals a novel proteolytic mechanism of receptoractivation. Cell 64: 1057–1068.

11. Hortin GL, Warshawsky I, Laude-Sharp M (2001) Macromolecular chromo-

genic substrates for measuring proteinase activity. Clin Chem 47: 215–222.12. Silphaduang U, Noga EJ (2001) Peptide antibiotics in mast cells of fish. Nature

414: 268–269.

13. Ainsworth DM, Erb HN, Eicker SW, Yeagar AE, Viel L, et al. (2000) Effects of

pulmonary abscesses on racing performance of horses treated at referral

veterinary medical teaching hospitals: 45 cases (1985–1997). J Am Vet MedAssoc 216: 1282–1287.

14. Smith TG, Robbins PA, Ratcliffe PJ (2008) The human side of hypoxia-inducible factor. Br J Haematol 141: 325–334.

15. Allen DH, Tracy PB (1995) Human coagulation factor V is activated to the

functional cofactor by elastase and cathepsin G expressed at the monocytesurface. J Biol Chem 270: 1408–1415.

16. Adams SA, Kelly SL, Kirsch RE, Shephard EG (1998) Digestion of 125I-labelled plasmin-derived fibrin degradation products by neutrophil lysosomal

enzymes. Blood Coagul Fibrinolysis 9: 307–313.17. Offermanns S, Laugwitz KL, Spicher K, Schultz G (1994) G proteins of the G12

family are activated via thromboxane A2 and thrombin receptors in human

platelets. Proc Natl Acad Sci U S A 91: 504–508.18. Matsumoto R, Sali A, Ghildyal N, Karplus M, Stevens RL (1995) Packaging of

proteases and proteoglycans in the granules of mast cells and otherhematopoietic cells. A cluster of histidines on mouse mast cell protease 7

regulates its binding to heparin serglycin proteoglycans. J Biol Chem 270:

19524–19531.19. Lentink D, Muijres FT, Donker-Duyvis FJ, van Leeuwen JL (2008) Vortex-wake

interactions of a flapping foil that models animal swimming and flight. J Exp Biol211: 267–273.

20. Muller UK, Stamhuis EJ, Videler JJ (2000) Hydrodynamics of unsteady fishswimming and the effects of body size: comparing the flow fields of fish larvae

and adults. J Exp Biol 203: 193–206.

21. Mahoney WC, Kurachi K, Hermodson MA (1980) Formation and dissociationof the covalent complexes between trypsin and two homologous inhibitors, alpha

1-antitrypsin and antithrombin III. Eur J Biochem 105: 545–552.



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